Project Summary Humans possess unique biological features compared to other primates. Among the most important of these are a larger and more complex cerebral cortex, and morphological changes in the limbs that allow humans to use sophisticated tools and walk upright. The evolution of these physical innovations has long been thought to involve sequence changes that altered gene regulation in development. However, the locations of such changes are only now beginning to be revealed, and their effects on gene expression and development remain unknown. The goal of this research project, which was initiated in 2010, is to identify regulatory elements with human-specific developmental activities and determine their biological impact. In the current funding period, we have focused on mapping sites likely to encode uniquely human promoter or enhancer functions using two complementary strategies. The first is experimental analysis of conserved noncoding sequences that show accelerated evolution in humans, which we and other groups have shown include developmental enhancers with human-specific activities. The second is comparative epigenetic analysis of limb and cortex development in human, rhesus macaque and mouse, to directly identify promoters and enhancers that have gained activity in humans. Sequences that exhibit both human-specific evolutionary acceleration and increased activity based on epigenetic marks are prime candidates for encoding novel regulatory functions with potentially large biological effects. Our priority in this renewal is to generate and study humanized mouse models for human accelerated regions that show epigenetic gains in the developing human limb or cortex. We will use CRISPR/Cas9 genome editing technology to rapidly generate genetically modified mice by gene targeting in single cell mouse embryos. We will then conduct genome-wide molecular analyses of embryonic development in these models to identify global changes in gene expression and regulation. Guided by the results of these studies, we will carry out targeted phenotypic analysis of limb and cortex development in humanized mice to identify biological processes altered by human-specific regulatory changes.